TLR LIGAND DECREASES MESENTERIC ISCHEMIA AND REPERFUSION INJURY-INDUCED GUT DAMAGE THROUGH TNF-α SIGNALING

Ischemic gut contributes to the development of sepsis and organ failure in critically ill patients. Toll-like receptors (TLRs) have been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We hypothesize that LIPS, a ligand for TLR4, decreases mesenteric I/R injury-induced gut damage through tumor necrosis factor alpha (TNF-alpha) signaling. First, wild-type (WT) mice were fed with oral antibiotics for 4 weeks to deplete the intestinal commensal microflora. At week 3, drinking water was supplemented with LPS (10 mu g/mu L) to trigger TLRs. The intestinal mucosa was harvested for TLR4 protein, caspase 3 activity, and terminal deoxynucleotide transferase labeling assay. Second, WT and Tnfrsf1a(-/-) mice received 30-min ischemia and 30-min reperfusion (301-30R) or 301-180R of the intestine; intestinal permeability and lipid peroxidation of the intestine were examined. Third, WT and Tnfrsf1a(-/-) mice were fed with oral antibiotics with or without LPS and received 301-180R of the intestine. The intestinal mucosa was harvested for lipid peroxidation; glutathione (GSH) level; nuclear factor kappa B (NF-kappa B) and AP-1 DNA-binding activity; Bcl-w, TNF-alpha, and CXCR2 mRNA expression; and HSP70 protein assay. Commensal depletion increased caspase 3 activity as well as villi apoptosis and decreased TLR4 expression of the intestinal mucosa. LIPS increased TLR4 expression and decreased villi apoptosis. Commensal depletion augmented 301-180R-induced intestine permeability as well as lipid peroxidation and decreased GSH level in WT mice but not in Tnfrsf1a(-/-) mice. LPS decreased 301-180R-induced intestinal permeability as well as lipid peroxidation and increased GSH level of the intestinal mucosa in WT mice but not in Tnfrsf1a(-/-) mice. Commensal depletion with 301-180R increased NF-kappa B and AP-1 DNA-binding activity, HSP70 protein expression, and decreased Bcl-w and TNF-alpha mRNA expression of the intestinal mucosa in WT mice but not in Tnfrsf1a(-/-) mice. Collectively, commensal microflora. induces TLR4 expression and decreases apoptosis of the intestinal mucosa. Commensal depletion enhances I/R-induced gut damage. LPS prevents I/R-induced intestinal permeability, lipid peroxidation, and decrease in GSH level. Given that the preventive effect of LPS on I/R-induced gut damage and NF-kappa B activity of the intestine is abolished in Tnfrsf1a(-/-) mice, we conclude that TLR ligand decreases mesenteric I/R injury-induced gut damage through TNF-alpha signaling.